The nuclear lamina is a meshwork of protein filaments that underlies the nucleoplasmic face of the inner nuclear membrane. This contiguous filamentous scaffolding forms at the nuclear periphery and provides structural support for the nucleus (Newport, 1990). The lamina plays a role in the regulation of gene expression through direct and indirect interactions with transcription factors (Ozaki, 1994). The lamina interacts directly with DNA and is also involved in chromatin organization via direct interactions with histones and other chromatin binding proteins (Gotzmann, 1999). Site-specific phosphorylation of lamina proteins results in the reversible disassembly of the lamina during mitosis (Haas, 1993), and these proteins are also targets of caspase cleavage during apoptosis (Slee, 2001).
The nuclear lamina is composed of intermediate filament proteins called lamins, which are subdivided into two families based on their expression profile and post-translational processing. Expression of the type-A lamins is timed to coincide with cellular differentiation, and expression of these proteins is considered a marker for embryonic stem cell differentiation (Constantinescu, 2006). Furthermore, while one of the A-type lamins, prelamin A, is post-translationally processed by the addition of a fifteen carbon farnesyl group, the covalent modification is later removed and mature A-type lamins lack this covalent modification. The B-type lamins are constitutively expressed in most cell types and are permanently post-translationally modified by the covalent addition of a hydrophobic fifteen carbon chain at the C-terminus, which results in their association with the nuclear membrane (Kitten, 1991). The A- and B-type lamins form heteropolymers and are the major constituents of the nuclear lamina.
Lamins are the oldest members of the intermediate filament family of proteins and, like other intermediate filament proteins, lamins contain a central alpha helical rod domain flanked by non-helical N-terminal and C-terminal domains (FIG. 1). Intercalation of the central alpha helical region is responsible for lamin dimerization, while the domains flanking the central region are involved in higher order lamin filament assembly. Phosphorylation of residues on either side of the alpha helical domain results in the reversible disassembly of the lamina during mitosis. The C-terminal domain of lamins contains a nuclear localization signal as well as a post-translational processing sequence. The two predominant type-A lamins, lamins A and C, are differentially transcribed from the same gene and lamin C differs from the other lamins in that it is essentially a truncated lamin lacking the post-translational processing sequence and containing a small unique C-terminus.
According to accepted theory, lamin C is not post-translationally processed, although other lamin proteins are post-translationally processed via a sequential series of covalent modifications (FIG. 2). Lamin proteins terminate in a CAAX motif (Cysteine, Aliphatic, Aliphatic, X-any), which is the target for post-translational farnesylation, the covalent addition of a fifteen carbon chain to the C-terminal cysteine residue. The farnesylated lamin protein becomes the substrate for an endoprotease which cleaves the protein on the C-terminal side of the modified cysteine residue, releasing the last three amino acids. The now C-terminal farnesylated cysteine residue is then further modified by a carboxymethyltransferase, which adds a methyl group to the end of the protein.
The carboxymethylation of the C-terminal cysteine residue is the final step in the processing of the B-type lamins, and the addition of the aliphatic carbon chain results in the association of the B-type lamins with the nuclear membrane, and the continued association of these proteins with membrane vesicles upon lamina breakdown and reassembly during mitosis. There are approximately twenty other human proteins which are known to also undergo farnesylation, and which are post-translationally processed by these same enzymes. The most noticeable of these proteins is the Ras gene product, the farnesylation of which also results in membrane association and protein activation.
While the post-translational processing of prelamin A proceeds through the same intermediates as the B-type lamins and other farnesylated proteins, prelamin A processing is unique among mammalian proteins as the final step in the maturation of lamin A is the endoproteolytic release of the remaining 15 C-terminal amino acid farnesylated and carboxymethylated peptide (FIG. 2). The function of prelamin A processing has been an enigma since it was first identified, as it seems to run against evolutionary conservation of energy in that extensive energy is used to post-translationally process the C-terminal portion of the peptide, which is subsequently thrown away. Early studies demonstrated that the prelamin A protein remains nucleoplasmic in mitotically arrested cells, and that mature lamin A can only incorporate into the nuclear lamina if the pre sequence is removed (Lutz, 1992; Izumi, 2000). However, if cells are allowed to cycle, the unprocessed prelamin A does incorporate into the lamina by hybridizing with already processed mature lamin A monomers, dimers or tetramers during lamina reassembly. As the previously incorporated phosphorylated mature lamin A protein monomers contain all the information necessary to properly localize and reform the nuclear lamina upon dephosphorylation, the post-translational processing of prelamin A as a method of protein targeting or assembly appeared redundant and no biological effect of replacing prelamin A with mature lamin A was observed in the mammalian cell lines studied.
The lamin A/C cDNA was first cloned and sequenced by Gunther Blobel in 1986 (Fisher, 1986), and early studies demonstrated these proteins were components of the nuclear lamina as well as the nucleoskeleton, the nuclear equivalent of the cellular cytoskeleton. Researchers interested in cholesterol metabolism studied prelamin A processing because the farnesyl group is generated in the cholesterol metabolic pathway. Cancer researchers testing farnesyltransferase inhibitors in Ras-related cancers used prelamin processing as a marker for drug inhibition of the farnesylation pathway.
In 1999, non-X-linked Emery Dreifuss muscular dystrophy (EDMD) was the first human disease identified as being associated with lamin A/C gene mutations (Bonne, 1999). As the X-linked form of EDMD is caused by mutations in emerin, a nuclear membrane protein that directly interacts with lamin A, it was not surprising that lamin A/C mutations could also cause this disease. While EDMD patients suffer from skeletal muscle and connective tissue abnormalities, their greatest heath risk is cardiovascular disease characterized by conduction defects, often necessitating pacemaker implantation.
Later the same year, lamin A/C mutations were also shown to be responsible for inherited forms of dilated cardiomyopathy characterized by conduction defects (Fatkin, 1999). In 2000, the present inventor and colleagues identified lamin A/C mutations as being responsible for dilated cardiomyopathy (DCM) and conduction defects in a family with variable skeletal muscle involvement (Brodsky, 2000). Some of the affected individuals had symptoms of EDMD, while others had indications of limb girdle muscular dystrophy, or no skeletal muscle involvement. Lamin A/C mutations were also shown to cause limb girdle muscular dystrophy, another disease characterized by DCM and conduction defects.
While all of these diseases affect skeletal and/or cardiac muscle, additional diseases were identified as being associated with lamin A/C mutations which did not share this phenotype. Lamin A/C mutations were shown to cause familial partial lipodystrophy, a fat storage disease in which patients typically develop insulin resistance and diabetes (Shackleton, 2000). Lamin A/C gene mutations were then shown to cause neurological and developmental disorders, including mandibuloacryl dysplasia (Novelli, 2002) and Charcot-Marie Tooth Syndrome (De Sandre-Giovannoli, 2002).
The distribution of lamin A/C mutations associated with the same disease as well as different diseases occur throughout the lamin A protein, suggesting that disruption of a particular structural or functional domain is not responsible for the different disease phenotypes. However, in the case of partial lipodystrophy, the mutations do cluster, and evidence has been presented suggesting some of the mutations may interfere with the binding of an adipocyte-specific transcription factor to lamin A (Lloyd, 2002).
Molecular studies of the mutant lamin proteins associated with diseases revealed that some cause obvious alterations in the nuclear lamina structure, with some mutations resulting in the formation of nuclear lamin aggregates (Raharjo, 2001) and/or changes in the cellular distribution of lamina-binding proteins (Mounkes, 2005). An increase in the percent of cells displaying nuclear herniations or “blebs” was also observed in cells expressing the mutant lamin constructs as compared to controls (Raharjo, 2001). While expression of some of the lamin A/C mutations were also shown to result in increased nuclear fragility and altered patterns of gene expression, an explanation for why different mutations affect different tissues and even different subgroups of the same tissue has not been identified. The finding that a lamin A/C knockout mouse shared many of the human disease pathologies (Sullivan, 1999) did not help to delineate why lamin A/C mutations have different tissue-specific effects.
Interest in lamin A/C increased tremendously when mutations in the gene were next found to be the sole cause of the premature aging syndrome Hutchinson-Gilford Progeria Syndrome (HGPS) (Eriksson, 2003). Patients with HGPS display postnatal growth retardation, midface hypoplasia, micrognathia, premature atherosclerosis and coronary artery disease, absence of subcutaneous fat, alopecia, generalized osteodysplasia with osteolysis and pathologic fractures, and the median age of death is 13 years of age. Unlike the other major progeroid syndrome, Werner's syndrome, HGPS is not associated with an increase in age-related cancers or cataracts, indicating that increased DNA mutability or decreased DNA repair is not responsible for the disease pathology as in Werner's syndrome. Instead, an apparent failure of post-natal tissue growth and repair mechanisms results in the striking appearance of premature aging and death.
A common silent mutation in lamin A/C gene resulting in the formation of a cryptic mRNA splice site and internal deletion at the C-terminal end of the prelamin A protein was found in the majority of HGPS patients (Eriksson, 2003). This mutation deletes the cleavage site necessary for the final prelamin A proteolytic processing step, and results in the partially processed prelamin A protein forming nuclear aggregates which result in increase in nuclear herniations, or “blebbing”.
Based on these findings, a commonly accepted model has been proposed in which prelamin A farnesylation targets the partially processed protein to the nuclear membrane where further processing releases the mature lamin A protein to incorporate into the nuclear lamina. In HGPS, the partially processed farnesylated prelamin A in the nuclear membrane causes nuclear blebbing, which is then proposed to cause all of the associated disease pathologies. However, nuclear blebbing has never been shown to be associated with any pathology seen in HGPS patients, or as interfering with any physiological process. Furthermore, an increase in nuclear blebbing results from expression of lamin A mutations which cause DCM and not HGPS. Nonetheless, drug trials have recently been reported in which farnesyltransferase inhibitors (FTIs) were used to inhibit prelamin aggregation and nuclear blebbing in vitro (Mallampalli, 2005; Toth, 2005; Yang, 2005), and in a mouse model of HGPS (Fong, 2006). However, while some improvement was observed when animals were treated with FTIs, a percentage of animals still displayed all of the disease phenotypes examined, and the authors conclude that blocking prelamin A processing would not cure the disease (Fong, 2006).
Therefore, there remains a need in the art to identify the molecular mechanisms responsible for the disease pathologies associated with lamin A/C mutations and/or dysfunction and to use the knowledge of the mechanisms to design therapeutic strategies for preventing and treating such diseases.